In recent years biopharmaceutical manufacturing has demonstrated major improvements in MAb production, exhibiting product titers as high as 25 g/L often associated with very high cell densities. High-density cell cultures with >150 million cells/mL pose a great challenge in further downstream processing because of a need to remove a large amount of biomass and increased levels of contaminants from cell debris generated during cell culture and harvesting. Production of biological substances (MAbs, in particular) usually involves processing a complex cell culture broth from which desired biological substances must be isolated and purified while maintaining high overall product recovery and quality. Traditionally, centrifugation and a combination of filtration techniques (tangential-flow filtration and depth filtration) have been widely accepted as workhorses for clarifying these complex cell culture broths. However, improvement of mammalian cell culture processes is providing for total cell densities far beyond traditional levels of 20×106 cells/mL for CHO cells to >150×106 cells/mL for PER.C6 cells. Thus, limitations of both centrifugation and filtration techniques are apparent by the high (≤40%) solids content of such harvests.
Centrifugation can be applied to process feed streams with high levels of solids, for instance. However, product recovery can be low because of increased pellet volumes and a need to desludge frequently (especially in large-scale continuous centrifugation). Additionally, cell disruption from shear forces generated during centrifugation can further decrease the efficiency of harvest clarification and potentially cause product damage and/or entrapment.
Depth filters are advantageous because they remove contaminants, and many come in single-use format, reducing the need for cleaning and validation. However, depth filters are currently unable to handle high-solids feedstreams and are often used in series with centrifugation. TFF can handle high solids loading, but this technique can exhibit poor yield because of polarization of solids at the membrane surface when processing highly dense feed streams. Excessive product dilution and cell lysis caused by shear forces can also limit the utility of TFF.
Flocculation of cell culture harvests has also been widely used to enhance clarification throughput and downstream filtration operations. Current techniques include the use of soluble polyionic polymers (such as DEAE dextran, acryl-based polymers, and polyethylene amine) and inorganic materials such as diatomaceous earth and perlites, which remove cells and cell debris. However, polymers must subsequently be removed from process streams, which requires monitoring and quantification by in-process and product-release assays. If IEX chromatography is included as a purification step in the downstream process, binding capacities will be greatly affected by the charged nature of flocculants. The high viscosity of polycation stock solutions presents an additional process challenge.
In recent years, various new clarification techniques have been developed. For example, (http://www.selectscience.net/product-news/sartorius-stedim-biotech) single use harvesting technology for high cell density cultures using diatomaceous earth (DE) as filter aid has been introduced. (Minow B., et al, BioProcess Int., Apr. 1, 2014). However, given its low surface area (BET˜1 m2/g) and non-porous nature, DE has no function in adsorption of proteins and/or other types of biologics, but only as filter aid.
In other processes, functionalized particulate materials have been used as adsorbents for clarification of cell broths. For example, WO2010043700 and WO2010043703 methods of clarification or removing host cell proteins by the utilization of anion exchange material in the process. The property or optimal conditions for the ion exchange materials are not described in the patent applications.
Efforts continue to further develop clarification materials and processes having improved agents in the form of functionalized particulate materials so as to improve binding capacity (i.e., the amount and the variety of biomaterials that can be bound to the adsorbent clarification agents) and/or binding selectivity of the functionalized particulate materials.